JP2011045389A - Composition for assaying glycated protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To precisely determine the ratio of glycated proteins and glycated albumin. <P>SOLUTION: There are provided compositions for accurately assaying a glycated protein by: (1) avoiding effects of globulin components and ascorbic acid, (2) stabilizing enzymes acting on protease and at least a glycated amino acid; (3) accurately assaying albumin; and (4) assaying glycated protein while avoiding the effects of glycated hemoglobin, and also an assay method. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a composition for measuring glycated protein and a method for measuring glycated protein. The composition for measuring glycated protein and the measuring method in the present invention are used for clinical examination, and glycated protein can be measured accurately.

In the diagnosis and management of diabetes, measurement of glycated protein is very important.
Glycated hemoglobin (GHb) that reflects the average blood glucose level of the month, glycated albumin (GA) that reflects the average blood glucose level for the past two weeks, and fructosamine (FRA), which is a generic term for glycated proteins that exhibit a reducing ability in serum, etc. Are measured on a daily basis. In GHb, the α-amino group of the β-chain N-terminal valine of hemoglobin is glycated, and in GA and FRA, albumin and the ε-amino group of the lysine residue of serum protein are glycated.

An enzyme method is an example of a highly accurate, simple and inexpensive method for measuring glycated protein. Examples thereof include Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, Patent Document 5, Patent Document 6, and the like.
However, in order to provide a composition that truly measures glycated protein accurately, it is essential to 1) avoid the effects of globulin components and ascorbic acid, 2) stabilize proteases, at least enzymes that act on glycated amino acids, When the glycated protein is glycated albumin,
3) A method for accurately measuring albumin, 4) It is also important to avoid the influence of glycated hemoglobin.

1) Prior art concerning avoidance of influence of globulin component and ascorbic acid It is known that globulin protein mass changes and affects FRA value in diabetic patients (for example, see Non-patent Document 1). Accordingly, the present inventors have developed a method for selectively inhibiting the action of protease on the globulin component by adding a certain metal ion or protein A or G to the protease reaction solution (see, for example, Patent Document 7). I have done it. In this invention, a glycated protein can be measured without being influenced by the globulin component, but as a globulin selective protease inhibitor used there, metal, protein A,
G is described. However, among the specific metals shown here, the strong metals are heavy metals, and it cannot be said that there are no environmental safety problems, and the weak metals are other reagent components (other compositions). ), The reagent solution may become cloudy. Proteins A and G are very expensive.

Furthermore, as a method of selectively adsorbing globulin in blood, a blood treatment agent that adsorbs endotoxin and globulin in blood using a chromatographic principle by introducing a ligand having a steroid skeleton into a vinyl copolymer. (For example, refer to Patent Document 8). However, according to the examination results in Table 1 of this Example, only α1 and α2 globulins have been confirmed to be adsorbed among globulins, and they do not show the adsorbing ability to γ globulins that account for 70% or more of the globulin components. In addition, even if γ globulin exhibits the ability to adsorb, it is unpredictable how much the protease can inhibit the action on γ globulin.

In recent years, ascorbic acid has been increasingly consumed in large quantities as a supplement, and clinical specimens containing high concentrations of ascorbic acid are increasing. Ascorbic acid causes various effects on clinical examinations due to its strong reducing power.
As a method for avoiding the influence of ascorbic acid in the sample, a method of eliminating ascorbic acid in the sample using a chemical method or an enzymatic method using ascorbate oxidase is known. When a glycated protein is fragmented using a protease and then the glycated amino acid produced using an enzyme that acts on at least the glycated amino acid is measured, the ascorbate oxidase (ASOx) is allowed to act simultaneously with the protease reaction, and the ascorbic acid is eliminated in advance. The method is preferable because it has little influence on the coloring system.

In the presence of protease, ascorbic acid in the test solution was erased using ASOx. For the test solution, 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) There is an example (see, for example, Non-Patent Document 2) that acts under the condition of buffer pH 8.0, and it is described that there is no change in the processing ability of ascorbic acid for 2 weeks in refrigerated storage.
However, as a result of studies by the present inventors, the ability to treat ascorbic acid in the presence of HEPES buffer pH 8.0, protease, and ASOx almost disappeared after storage at 37 ° C for 1 day or 10 ° C for 2 weeks.

2) Conventional technology related to stabilization of proteases and enzymes that act at least on glycated amino acids For clinical measurement of glycated proteins, proteases in a high concentration are used in an aqueous solution that is unthinkable in other fields such as the food field. Is done. Proteases are known to undergo self-digestion in aqueous solutions and are unlikely to exist stably in such high-concentration aqueous solutions.
Therefore, the protease used for the composition for measuring glycated protein so far is supplied as a freeze-dried product.
There is no example in which the protease is stabilized to a level that can be stored in a liquid form for a long period of time with respect to the composition and method for measuring glycated protein. Similarly, there is no example in which the enzyme acting on glycated amino acid is stabilized at least to a level that can be stored in a liquid state for a long period of time with respect to the glycated protein measuring composition and measuring method.

3) Conventional technology for accurately measuring albumin As an albumin measurement method, an immunization method using an anti-albumin antibody, bromocresol green (
BCG), dye method using bromocresol purple (BCP) and the like. The dye method is widely used in daily examinations because of its simple operation and low cost, but the BCG method has a drawback that its action on the globulin component has been confirmed and its specificity for albumin is low.
On the other hand, the BCP method is highly specific for albumin, but is easily affected by coexisting substances.
In addition, there is a problem that the value varies depending on the redox state of albumin due to the influence of the SH compound. As a method for solving this problem, an example in which a BCP reaction is performed in the presence of a protein denaturant and / or an SH reagent (for example, see Patent Document 9) is known. However, there have been no studies on BCP reactivity to GA and non-glycated albumin (NGA).

4) Conventional technology for avoiding the influence of glycated hemoglobin As described above, GA has ε-amino group of albumin glycated, while GHb has glycated α-amino group of β-chain N-terminal valine in hemoglobin. Therefore, when GA is used as a measurement target, it is preferable that only amino acids in which the ε-amino group is glycated can be measured.

There have been some known enzymes that show high specificity for ε-amino groups and do not act on glycated valine (see, for example, Patent Document 10), but none are supplied at a cost that is practically practical. . Of these, fructosyl amino acid oxidase (FOD) derived from Fusarium oxysporum is particularly useful because of its high reactivity, and the inventors have isolated and reported the FOD gene (see, for example, Patent Document 11). However, the production method using this gene is highly productive and can produce FOD at a low cost, but it has been confirmed to be reactive to glycated valine with α-amino group saccharified, which is satisfactory in terms of specificity. It wasn't.

JP-A-6-46846 Japanese Unexamined Patent Publication No. 5-192193 Japanese Unexamined Patent Publication No. 2-195900 Japanese Patent Laid-Open No. 2-195899 WO98 / 48043 Publication WO97 / 13872 Publication Japanese Patent Application No. 11-231259 Japanese Patent Laid-Open No. 61-94663 Japanese Patent Laid-Open No. 10-232233 JP 11-243950 A Japanese Patent Laid-Open No. 10-201473

Rodrigues S et al, Clin Chem. 35: 134-138 (1989) Clinical Chemistry 27: 99-106,1998

An object of the present invention is to provide a composition and a stabilization method in which 1) the effects of globulin components and ascorbic acid are avoided, and 2) the protease and at least the enzyme that acts on glycated amino acids are stabilized in accurately measuring glycated protein. Furthermore, when the glycated protein is glycated albumin, 3) to accurately measure albumin, and 4) to provide a composition for avoiding the influence of glycated hemoglobin and a method for avoiding the influence.

In accurately measuring glycated proteins, 1) avoiding the effects of globulin components and ascorbic acid, 2) stabilization of proteases and enzymes that act at least on glycated amino acids are essential for accurate measurement of glycated proteins. 3 is glycated albumin
) Accurate measurement of albumin, 4) Avoiding the effects of glycated hemoglobin is essential.

1) Avoidance of influence of globulin component and ascorbic acid As a result of intensive studies, the present inventors have determined that a protease reaction solution contains deoxycholic acid, deoxycholic acid amide, cholic acid amide, quaternary ammonium salt or quaternary ammonium salt type positive Addition of one or more selected from ionic surfactant, concanavalin A, octyl glucoside or betaine can selectively inhibit the action of protease on the globulin component, and the enzyme which acts on this reaction solution at least on glycated amino acids The present inventors have found that glycated proteins can be measured easily and accurately with no reproducibility even if they are directly acted on, with good reproducibility. Moreover, these compounds are economically advantageous, have no environmental safety problems as compared with the case of using conventional techniques, and do not cause turbidity when mixed with a specimen.

In addition, it is efficient to use ASOx to eliminate ascorbic acid, but it is usually difficult to think that ASOx is stably present in a reaction solution containing a large amount of protease. In fact, as a result of examination by the present inventors, conditions for allowing ASOx to stably exist in a reaction solution containing a large amount of protease were not found in the examination of the type of protease, protease inhibitor, and type of ASOx.
However, as a result of intensive studies, the present inventors have unexpectedly found that the stability of ASOx is dramatically improved depending on the type of buffer solution.

2) Stabilization of proteases and enzymes that act at least on glycated amino acids For clinical measurement of glycated proteins, a high concentration of protease is used in the form of an aqueous solution as described above, and the protease itself is basically unstable. However, as a result of intensive studies, the present inventors have added dimethyl sulfoxide, alcohol, water-soluble calcium salt, sodium chloride, quaternary ammonium salt or quaternary ammonium salt type cationic surfactant, so that It has been found that the stability is dramatically improved and that it can be stored for a long time even under liquid and high concentration conditions.

Moreover, at least the enzyme that acts on glycated amino acids is an enzyme whose activity decreases to about 10% when stored at 37 ° C. for 4 days in a liquid state, and is not a stable enzyme. However, as a result of intensive studies, the present inventors have added a stabilizer selected from sugar alcohol, sucrose, water-soluble magnesium salt, water-soluble calcium salt, ammonium sulfate, amino acid, and sarcosine to at least an enzyme that acts on a glycated amino acid. It has been found that a surprising stabilization effect is obtained such that almost no decrease in activity is observed when stored in liquid form at 37 ° C. for 4 days.

In addition, proteases exhibit strong proteolytic activity near the optimum pH, but at the same time, self-digestion proceeds, making it difficult to store in liquid form. However, as a result of intensive studies, the present inventors have made the first reagent suitable for protease action, and formulated the second reagent under the stable condition even in liquid form, so that it does not affect the measurement conditions. It was found that the protease can be stably stored, and surprisingly, even if the enzyme used for the pretreatment reaction is formulated in the second reagent, the measurement can be accurately performed without affecting the measurement. In addition, by prescribing at least an enzyme that acts on a glycated amino acid in the first reagent, it was possible to erase the glycated amino acid present in the sample in advance and selectively measure the glycated protein.

3) Method for accurately measuring albumin Surprisingly, the inventors have found that the reactivity of BCP differs between GA and NGA, and that when NGA is mixed in a large amount, the value is negatively affected. Therefore, as a result of intensive studies, the present inventors have successfully treated albumin containing a large amount of NGA accurately by treating the sample with a protein denaturant and / or a compound having an SS bond before or simultaneously with albumin measurement. It was found that it can be measured.

4) Avoidance of the influence of glycated hemoglobin The present inventors have conducted intensive research on the above problems, and as a result, Fusarium oxysporum IFO-99.
By modifying the FOD gene from 72 strains to create mutant FOD and measuring its properties, N
Substitution of the 372nd lysine from the terminal with other amino acids found that the substrate specificity was remarkably changed, and the reactivity to glycated valine was remarkably low. Created.
This mutant FOD was made based on the above findings, and is a mutant FOD in which glycated valine reactivity disappeared by substituting lysine of amino acid sequence 372 of SEQ ID NO: 1 with another amino acid for FOD. is there. The mutant FOD according to claim 1, wherein the lysine of amino acid sequence 372 of SEQ ID NO: 1 is substituted with tryptophan, methionine or valine.
Finally, the inventors have completed these compositions and completed a composition and measurement method for accurately measuring glycated protein.

According to the present invention, it has become possible to more accurately measure the ratio of glycated protein and glycated albumin in a subject. Therefore, it can be usefully used as a clinical test drug.

Hereinafter, the configuration and preferred embodiments of the present invention will be described in more detail.
As the protease that can be used in the present invention, any protease can be used as long as it effectively acts on a glycated protein contained in a test solution and effectively produces a glycated amino acid and / or glycated peptide derived from the protein. Well, for example, animals, plants, genus Bacillus, Aspergillus, Rhizopus, Penicillium, Streptomyces, Staphylococcus, Clostridium
Examples include proteases derived from microorganisms such as ridium, Lysobacter, Glifila, Yeast, Tritirachium, Thermus, Pseudomonus, and Achromobacter.

In addition, when GA is the glycated protein to be measured, protease derived from microorganisms of the genus Bacillus and Streptomyces is preferred because it has a large effect on human albumin (Alb). In addition, when the glycated protein to be measured is GHb, the genus Bacillus, Aspergillus,
Proteases from the genus Streptomyces and Trityratium are human hemoglobin (Hb)
It is more preferable because of its large effect on.
A method for measuring the activity of a protease that can be used in the present invention is shown below.

<< Method for measuring protease activity >>
The protease activity showing a color corresponding to 1 μg of tyrosine per minute at 30 ° C. under the following measurement conditions is expressed as 1 PU (proteolytic Unit).
<Substrate> 0.6% milk casein (Merck)
<Enzyme solution> Dilute to 10 PU to 20 PU <Enzyme dilute solution> 20 mM acetate buffer pH 7.5
1mM calcium acetate
100 mM sodium chloride <reaction stop solution> 0.11M trichloroacetic acid
0.22M sodium acetate
0.33M Acetic acid <Operation>
Dissolve the protease solution in an enzyme dilution solution so as to be 10 to 20 PU / ml, take 1 ml of this solution in a test tube and warm to 30 ° C. Add 5 ml of substrate solution preheated to 30 ° C, and after 10 minutes, add 5 ml of stop solution to stop the reaction. The mixture is heated at 30 ° C. for 30 minutes as it is to coagulate the precipitate, and filtered with Toyo filter paper N0.131 (9 cm) to obtain a filtrate. In blank measurement, 1 ml of protease solution is placed in a test tube and heated to 30 ° C. First, 5 ml of stop solution is added, followed by 5 ml of substrate solution.
Aggregate and filter in the same way after adding l. Add 2 ml of the filtrate to 5 ml of 0.55 M sodium carbonate solution and 1 ml of 3-fold diluted forin reagent, and react at 30 ° C for 30 minutes, and then measure the absorbance at 660 nm. The absorbance change obtained by subtracting the absorbance of the blank measurement from the absorbance at which the enzyme action was performed is obtained, and the enzyme activity is obtained from a separately prepared action standard curve.

<Standard action curve creation method>
Dilute the enzyme solution adjusted to about 50 PU / ml to prepare an enzyme solution with a series of dilution ratios of 2 to 50 PU / ml and perform the above operation.The resulting absorbance change is plotted on the vertical axis and the dilution factor is plotted on the horizontal axis. Plot. On the other hand, L-tyrosine is dissolved in 0.2N hydrochloric acid at a concentration of 0.01%, and 1 ml of this is added with 10 ml of 0.2N hydrochloric acid to obtain a standard tyrosine solution (tyrosine concentration: 9.09 μg / ml). Standard tyrosine solution 2ml and 0.2
Perform the above measurement procedure for 2 ml of N hydrochloric acid, and the absorbance change obtained was 18.2 μg of tyrosine.
It corresponds to. The change in absorbance is plotted on the graph, and a perpendicular line is drawn from the point to the horizontal axis, and the intersection with the horizontal axis corresponds to 10 PU / ml.

In addition, the use concentration of these proteases is not particularly limited as long as the target protein can be efficiently cleaved within a certain period of time, for example, usually 1 to 100,000 PU / ml, preferably 10 to 10,000 P
It may be used at a concentration of U / ml.
As an enzyme that acts on at least a glycated amino acid that can be used in the present invention, it effectively acts on a glycated amino acid or a glycated peptide produced from a glycated protein contained in a test solution by the action of the protease, and substantially glycated protein. Any enzyme can be used as long as it can measure, but at least an enzyme that acts on a glycated amino acid that works well on an amino acid with an α-amino group, and at least a glycation that works on an amino acid with an ε-amino group on it. Examples include enzymes that act on amino acids.
Examples of enzymes that act at least on glycated amino acids whose ε-amino group works well on glycated amino acids include the genus Gibberella, the genus Aspergillus, the genus Candida, the genus Penicillium, Examples include FOD derived from the genus Fusarium, the genus Acremonium, or the genus Debaryomyces.
An example of an enzyme that acts on at least a glycated amino acid that acts well on an amino acid whose α-amino group is glycated is an enzyme derived from Corynebacterium.

Furthermore, examples of enzymes that have sufficient activity even in the presence of proteases and can be produced at low cost include genetically modified ketoamine oxidase (R-FOD; manufactured by Asahi Kasei Co., Ltd.) and, in addition, glycated valine reactivity. A mutant FOD (R-FOD-II; manufactured by Asahi Kasei Co., Ltd.) that significantly reduces
The DNA encoding the FOD protein derived from Fusarium oxysporum IFO-9972, which is the source of R-FOD-II, is extracted by chromosomal DNA from Fusarium oxysporum IFO-9972 by conventional methods, followed by PCR and hybridization methods. It can be obtained by separating with the above.
For introducing the mutation into the obtained FOD gene, the PCR method may be applied or a site-specific mutation method may be used if a mutation is directly added to DNA. If the mutation is due to random mutation, an E. coli host having a reduced DNA repair ability may be used, or a host organism into which the FOD gene has been introduced can be cultured in a medium supplemented with a DNA mutation source.

The thus obtained mutant FOD gene is introduced into a host microorganism using an appropriate host-vector system, and screening is performed using an expression vector marker and FOD activity expression or DNA probe as an index, and a recombinant containing the FOD gene. Isolate the microorganism holding the DNA plasmid,
Mutant FO is obtained by culturing the genetically modified microorganism and extracting and purifying the recombinant protein from the cultured cells.
D can be obtained.

In order to obtain mutant FOD, specifically, it may be carried out as follows. Among the operating methods, those which are conventionally used are, for example, the method of Maniatis et al. (Maniatis, T., et al. Molecular Clon
ing. Cold Spring Harbor Laboratory 1982, 1989), and various procedures for attaching various commercially available enzymes and kits.
To introduce mutations into the isolated FOD gene, PCR using Taq polymerase and other 3 '→ 5' repair-deficient polymerases under the condition of manganese ion addition, and E. coli hosts lacking DNA repair ability It is only necessary to induce a gene mutation by culturing in a medium in which a FOD gene is introduced and a mutation source such as dianisidine is added, and to isolate a mutant that has acquired the desired substrate specificity from the prepared mutation candidate group.
The FOD mutation introduced by the above-described method is the dideoxy method (Sangar, F. (1981) Scienc
e, 214, 1205-1210) by confirming the base sequence of the gene into which the mutation has been introduced.
Once mutations have been determined, Zoller's method (Zoller, MJ and Smith, M. (1983)
Specific mutations can also be introduced by site-specific mutagenesis according to Methods in Enzymology, 154, 367).

The mutation of the amino acid sequence of the polypeptide constituting the mutant FOD can be determined from the base sequence of the mutant gene. The mutant FOD obtained by the above method can be produced as a recombinant by incorporating the mutant FOD gene into an appropriate host-vector system.
As a vector into which a mutant FOD gene is incorporated, a vector constructed for genetic recombination from a phage or plasmid that can autonomously grow in a host microorganism is suitable. For example, a microorganism belonging to E. coli can be used as a phage vector. Λgt for host microorganism
• λC, λgt, λB, etc. can be used. Examples of plasmid vectors include E.c.
When using oli as a host microorganism, plasmids pBR322, pBR325, pACYC184, pUC12, pUC13, p
UC18, pUC19, pUC118, pIN I, BluescriptKS +, pUB110, pKH when using Bacillus subtilis
300PLK, pIJ680 and pIJ702 can be used when actinomycetes are used as hosts, and Yrp7, pYC1 and Yep13 can be used when yeasts, particularly Saccharomyces cerevisiae, are used as hosts.
In order to incorporate the mutant FOD gene into such a vector, both are cleaved with an appropriate restriction enzyme that produces the same end, and the DNA fragment containing the mutant FOD gene and the vector fragment are ligated together by a DNA ligase enzyme according to a conventional method. Just do it.

The host microorganism into which the vector to which the mutated FOD gene is bound may be transferred as long as the recombinant DNA can be stably and autonomously propagated.For example, when the host microorganism belongs to E. coli, E. coli DH1, E. E. coli JM109, E. coli W3110, E. coli C600, etc. can be used. In addition, when the microbial host is a microorganism belonging to Bacillus subtilis, Bacillus subtilis (Bacillus subtilis)
In the case of microorganisms belonging to actinomycetes, such as ISW1214, Streptomyces lividans (Strepto
For microorganisms belonging to Saccharomyces cerevisiae, such as myces lividans) TK24, Saccharomyces cerevisiae INVSC1 can be used.

As a method for transferring the recombinant DNA into the host microorganism, for example, when the host microorganism belongs to E. coli, Saccharomyces cerevisiae, or Streptomyces lividans, it is recombined into a competent cell transformed host strain according to a conventional method. Just transfer the DNA,
Depending on the strain, electroporation may be used.
In order to produce mutant FOD, the host into which the mutant FOD gene has been introduced is cultured in an appropriate medium, and the cultured cells are collected and then subjected to ultrasonic disruption or lysozyme treatment in an appropriate buffer. And the bacterial cell extract may be prepared. Alternatively, the mutant FOD may be accumulated in the culture solution by secretory expression by adding a signal sequence.
The mutant FOD produced in this way is separated and purified by ordinary ammonium sulfate precipitation, gel filtration, column purification, etc., and supplied as an enzyme preparation.

The quantitative relationship generally used for the above-described genetic manipulation is that about 0.1 to 10 μg of DNA and vector DNA from the donor microorganism, about 1 to 10 U of restriction enzyme, about 300 U of ligase, and about 1 to 10 of other enzymes.
U, degree is illustrated.
As a specific example of a transformed microorganism containing a mutant FOD gene and capable of producing a mutant FOD,
Escherichia coli JM109 / pcmFOD3, a transformed microorganism having a plasmid pcmFOD3 containing a mutated FOD gene as a host microorganism and belonging to Escherichia coli
(FERM BP-7847), and a transformed microorganism carrying pcmFOD4, Escherichia coli JM109
・ Transformed microorganisms carrying pcmFOD4 and pcmFOD5, Escherichia coli JM109 ・ pcmFO
D5 (FERM BP-7848). These structures are shown in FIG.
In addition, Escherichia coli JM109 / pcmFOD3 and Escherichia coli JM109 / pcmFOD5
Both were deposited internationally at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center, 1st Street, 1st Street, Tsukuba City, Ibaraki Prefecture, Japan, on January 16, 2001.
47 and FERM BP-7848.

In producing the mutant FOD using the transformed microorganism, the transformed microorganism is cultured in a nutrient medium to produce the mutant FOD in the cells or in the culture solution. The cells are collected by means such as filtration or centrifugation, and then the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, EDTA and / or an appropriate surfactant are added. Add and concentrate the aqueous solution of the mutant FOD, or treat it by adsorption chromatography such as ammonium sulfate fractionation, gel filtration, affinity chromatography, or ion exchange chromatography without concentrating it, Obtainable.

The culture conditions of the transformed microorganisms may be selected in consideration of the nutritional physiological properties. Usually, in most cases, liquid culture is used. is there. As a nutrient source of the medium, those commonly used for culturing microorganisms can be widely used.
As the carbon source, any carbon compound that can be assimilated may be used. For example, glucose, saccharose, lactose, maltose, fructose, molasses and the like are used. The nitrogen source may be any available nitrogen compound, and for example, peptone, meat extract, yeast extract, casein hydrolyzate and the like are used.
In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

The culture temperature can be appropriately changed within the range in which microorganisms grow and produce mutant FOD, but in the case of E. coli, it is preferably about 20 to 42 ° C. Culture conditions vary slightly depending on conditions, but mutant FOD
The cultivation may be terminated at an appropriate time in anticipation of reaching the maximum end of E. coli. In the case of E. coli, it is usually about 12 to 48 hours. The pH of the medium can be appropriately changed within the range in which the bacteria grow and produce mutant FOD, but in the case of E. coli, it is preferably about pH 6-8.

The mutant FOD in the culture can be collected and used as it is in the culture solution containing the bacterial cells, but generally, when the mutant FOD is present in the culture solution, filtration, centrifugation, etc. It is used after separating the mutant FOD-containing solution and the microbial cells. When the mutant FOD is present in the microbial cells, the microbial cells are collected by means such as filtration or centrifugation of the obtained culture,
Next, the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme as necessary, and a chelating agent such as EDTA and / or a surfactant is added to solubilize the mutant FOD and separate and collect it as an aqueous solution. .
The mutant FOD-containing solution thus obtained is, for example, concentrated under reduced pressure, membrane concentrated, further ammonium sulfate,
What is necessary is just to precipitate by the fractional precipitation method by salting-out processing, such as sodium sulfate.

The precipitate can then be dissolved in water and dialyzed through a semipermeable membrane to remove lower molecular weight impurities. In addition, purification by gel filtration using an adsorbent or gel filtration agent, adsorption chromatography such as affinity chromatography, ion exchange chromatography, etc., from a mutant FOD-containing solution obtained by using these means, vacuum concentration freeze-drying, etc. A purified mutant FOD can be obtained by the above treatment.
The activity of the enzyme acting on the glycated amino acid was measured by the following method.

<< Method for measuring activity of enzyme acting on glycated amino acid >>
<Composition of reaction solution>
50 mM Tris buffer pH 7.5
0.03% 4-aminoantipyrine (4-AA) (Wako Pure Chemical Industries, Ltd.)
0.02% Phenol (Wako Pure Chemical Industries)
4.5U / ml peroxidase (POD) (Sigma)
1.0 mM α-carbobenzoxy-ε-D-fructosyl-L-lysine or fruct
Silvaline (synthesized and purified based on the method of Hashiba et al. Hashiba H,
J.Agric.Food Chem.24: 70, 1976 The following are abbreviated as ZFL and FV. )
Place 1 ml of the above reaction solution in a small test tube, preheat at 37 ° C. for 5 minutes, add 0.02 ml of an appropriately diluted enzyme solution, and stir to start the reaction. 2 ml of 0.5% SDS after exactly 10 minutes of reaction
The reaction is stopped by addition, and the absorbance at a wavelength of 500 nm is measured (As). Further, the absorbance is measured by performing the same operation using 0.02 ml of distilled water instead of the enzyme solution as a blank (Ab). The enzyme activity is determined from the absorbance difference (As-Ab) between the absorbance of this enzyme action (As) and the absorbance of the blind (Ab). Separately, using a standard solution of hydrogen peroxide, the relationship between the absorbance and the generated hydrogen peroxide was examined,
-1 The amount of enzyme that produces 1 μmol of hydrogen peroxide per minute is defined as 1U. The calculation formula is shown below.
3.02: Total reaction volume (ml)
0.02: Enzyme solution volume (ml)
10: Reaction time
2: Dye 1 with 4-AA and phenol condensed from 2 molecules of hydrogen peroxide
Factor due to generating numerator
12.0: millimolar extinction coefficient of 4-AA-phenol
B: Among mutant FODs obtained by a method more than the dilution ratio of the enzyme solution, the enzymatic properties of the mutant FOD in which the lysine of amino acid sequence 372 of SEQ ID NO: 1 was replaced with tryptophan are as follows.

(1) Substrate specificity
ZFL 100%
FV 0%
(2) Enzyme action As shown below, it catalyzes a reaction that decomposes at least an α-amino acid and ε-amino acid Amadori compound to produce glucosone, hydrogen peroxide, and the corresponding α-amino acid and ε-amino acid.
(3) Molecular weight The molecular weight of this enzyme is 0.1M containing 0.2M NaCl by column gel filtration using Sephadex · G-100.
As a result of measuring the phosphate buffer solution (pH 7.0) of 48000 ± 2000, SDS-PAGE was 4700.
0 ± 2000.
(4) Isoelectric point After applying electricity for 40 hours at 4 ° C. and a constant voltage of 700 V by a focused electrophoresis method using carrier ampholite, fractionation was performed, and the enzyme activity of each fraction was measured. As a result, pH 4.3 ± 0.2 Met.
(5) Km value
50 mM Tris-HCl buffer (pH 7.5)
0.03% 4-AA
0.02% phenol
As a result of measuring the Km value for ZFL by changing the concentration of the synthetic substrate ZFL in a reaction solution containing 4.5 U / ml peroxidase, 3.
A value of 4 mM was shown.
(6) Optimum pH
According to the enzyme activity measurement method described above, instead of 50 mM Tris-HCl buffer (pH 7.5) in the reaction solution, 100 mM acetate buffer (pH 4.4-5.4), phosphate buffer (pH 5.6-7.9) ), Tris-HCl buffer (pH 7.3-8.5), and glycine-sodium hydroxide buffer (pH 8.0-10.3). As a result, the maximum activity was shown at pH 7.5.
(7) pH stability 0.5 ml of various buffers used for measuring the optimum pH of 0.5 M containing 0.5 U of this enzyme at 40 ° C., 1
After treatment for 0 minute, the residual activity was measured according to the activity measurement method described below. As a result, pH 7.0
It retained 80% or more of activity in the -9.0 range.
(8) Thermostability 0.5 U of this enzyme was prepared with 0.2 M Tris-HCl buffer (pH 7.5), and after 10 minutes of heat treatment, the residual activity was measured according to the activity measurement method. As a result, the residual activity was maintained at 95% or higher up to 40 ° C.
(9) Optimal temperature
Using 40 mM Tris-HCl buffer (pH 7.5), according to the activity measurement method, after reaction at each temperature for 10 minutes, the reaction was stopped with 2 ml of 0.5% sodium lauryl sulfate (hereinafter abbreviated as SDS), and the wavelength was reduced. Absorbance was measured at 500 nm. As a result, the maximum activity was shown at 50 ° C.

Next, the glycated lysine in the test solution is erased by using a mutant FOD that significantly reduces the glycated valine reactivity by substituting lysine of amino acid sequence 372 of the sequence listing SEQ ID NO: 1 with other amino acids. After that, a method for measuring glycated valine in a test solution using FOD reactive to glycated valine will be described.
The FOD that eliminates the glycated lysine in the test solution is not limited as long as it is a FOD that does not act on glycated valine. For example, FOD uses lysine in the amino acid sequence of SEQ ID NO: 1 in the sequence listing as another amino acid. Mutant FOD significantly reduced glycated valine reactivity by substitution
More preferably, among the mutant FOD, a mutant FOD in which the lysine of amino acid sequence 372 of SEQ ID NO: 1 is substituted with tryptophan, methionine, or valine may be used. At this time, the amount of the enzyme added to the reaction solution may be an amount sufficient to eliminate glycated lysine in the test solution, and is, for example, 0.5 to 200 U / ml, preferably 1 to 50 U / ml.
The FOD for measuring glycated valine is not limited as long as it is FOD reactive to glycated valine. For example, FOD derived from Fusarium oxysporum IFO-9972 may be used. The amount of enzyme added to the reaction solution at this time may be an amount sufficient to measure glycated valine in the test solution, and is, for example, 0.5 to 200 U / ml, preferably 1 to 50 U / ml.

Specifically, first, in the first reaction, mutant FOD is allowed to act on glycated lysine in a test solution containing glycated lysine and glycated valine, and the hydrogen peroxide produced at this time is decomposed by catalase or the like. In the second reaction, hydrogen peroxide produced by the action of FOD on glycated valine in the test solution is reacted with 4-aminoantipyrine (4-AA) and a Trinder reagent, and the resulting dye is colorimetrically measured. That's fine. Further, sodium azide which is an inhibitor of catalase may be added to the second reaction solution.

As a globulin component-selective protease inhibitor that can be used to accurately measure glycated protein using the present invention, a protease is allowed to act on a test solution in the presence of a globulin component-selective protease inhibitor. Any inhibitor may be used as long as it is a globulin component selective inhibitor capable of fragmenting a protein other than the globulin component. Preferred examples thereof include deoxycholic acid, deoxycholic acid amide, cholic acid amide, quaternary ammonium salts,
Quaternary ammonium salt type cationic surfactants, concanavalin A, octyl glucoside or betaine are listed.
As the deoxycholic acid amide, for example, bisgluconamidopropyl deoxycholamide (N, N-Bis (3-D-gluconamidopropyl) deoxycholamido) and the like are preferable, and as the cholic acid amide, for example, sulfate-3-[(coal Amidopropyl) dimethylammonio] -1-propane (3-[(3
-Cholamidopropyl) dimethylammonio] -2-hydroxypropanesulfonic acid), 3-[(cholamidopropyl) dimethylammonio] -2-hydroxy-1-propane (3-[(3-Cholamidoprop
yl) dimethylammonio] propanesulfonic acid) or bisgluconamidopropyl cholamido (N, N-Bis (3-D-gluconamidopropyl) cholamido) or the like is preferable.
As the quaternary ammonium salt, for example, benzyltriethylammonium chloride, benzyltri-n-butylammonium chloride and the like are preferable, and as the quaternary ammonium salt type cationic surfactant, for example, lauryltrimethylammonium chloride or lauryldimethylamine oxide, etc. Is preferred.
These globulin component selective inhibitors may be used alone or in combination.

In addition, the use concentration of these globulin component-selective inhibitors may be an amount that can sufficiently suppress the action on the globulin component during the protease action. For example, deoxycholate, deoxycholate, cholate amide In the case of using octylglucoside, quaternary ammonium salt or quaternary ammonium salt type cationic surfactant, it is preferably used at a concentration of about 0.01% to 20%, more preferably 0.05% to 10%. In addition, other concentrations can be used.
Similarly, for example, when using concanavalin A, octyl glucoside or betaine, they can be used at concentrations of 0.01 to 10 mg / ml and 0.005 to 5%, respectively.
It is preferable to use at a concentration of 0.02 to 2 mg / ml, 0.01 to 2%, and other concentrations can be used.

As ASOx that can be used when accurately measuring glycated protein using the present invention, any ASOx that effectively acts on ascorbic acid contained in a test solution may be used. Examples include microorganism-derived ASOx. Although a specific example is shown below, these are only examples and are not limited at all.
Examples of plant-derived ASOx include cucumber (Amano, Toyobo) and pumpkin (Roche, Toyobo).
Examples of ASOx derived from microorganisms include those derived from Acremonium (Asahi Kasei) and microorganisms (Amano).
The activity of ASOx was measured by the following method.

<< ASOx activity measurement method >>
<Storage substrate solution>
L ascorbic acid (Wako Pure Chemical Industries, Ltd.) 176 mg and EDTA (Daiichi Chemicals Co., Ltd.) 37 mg, 1 mM hydrochloric acid 100
Dissolve in ml.
<Reaction reagent mixture>
The above stock substrate solution was washed with 90 mM 2 potassium phosphate-5 mM 1 phosphate phosphate buffer containing 0.45 mM EDTA.
Dilute twice.
<Operation>
Put 1 ml of the above reaction reagent mixture in a small test tube, preheat it at 30 ° C. for 5 minutes, add 0.10 ml of an appropriately diluted enzyme solution and stir to start the reaction. After the reaction for exactly 5 minutes, 3.0 ml of 0.2N hydrochloric acid aqueous solution is added to stop the reaction, and the absorbance at a wavelength of 245 nm is measured (As). Also, 1 ml of the above reaction solution as a blank is put into a small test tube, preheated at 30 ° C. for 5 minutes, and then 0.2N aqueous hydrochloric acid solution 3
Stop the reaction by adding .0 ml. Add 0.10 ml of an appropriately diluted enzyme solution and stir to a wavelength of 24
Measure absorbance at 5 nm (Ab). Absorbance difference between the absorbance of this enzyme action (As) and the absorbance of the blind (Ab) (
Obtain enzyme activity from Ab-As). The amount of enzyme that oxidizes 1 μmol of ascorbic acid to dehydroascorbic acid at 30 ° C. for 1 minute is defined as 1 U. The calculation formula is shown below.
10.0: millimolar extinction coefficient of ascorbic acid at 245 nm under the condition of pH 1.0
5: Reaction time (min)
4.10: Total reaction volume (ml)
0.10: Amount of enzyme sample solution used for reaction B: Dilution ratio of enzyme solution In addition, the concentration of these ASOx is sufficient to eliminate ascorbic acid even when using reagents when storing in the presence of protease and ASOx. Any amount is possible, for example, usually 0.
The concentration may be 1 U / ml to 100 U / ml, preferably 1 U / ml to 50 U / ml.

4- (2-) that can be used in combination with ASOx in accurately measuring glycated proteins using the present invention.
As the buffer having no hydroxyethyl) -1-piperazinyl group, any buffer can be used as long as ASOx is stable in the presence of protease and ASOx. For example, 3- [4 -(2-Hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS), 2- [4- (2
-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) and 2-hydroxy-3- [4
-(2-Hydroxyethyl) -1-piperazinyl] propanesulfonic acid (HEPPSO) and other buffering agents other than those having a 4- (2-hydroxyethyl) -1-piperazinyl group can be used. Also good.
Further, examples of preferable buffering agents include, for example, N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N- (2-acetamido) iminodiacetic acid (ADA), N, N-bis (2- Hydroxyethyl
) -2-Aminoethanesulfonic acid (BES), N, N-bis (2-hydroxyethyl) glycine (Bicine),
Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO) N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-
[N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), 2 -Hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis (2-ethanesulfonic acid (PIPES), piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) (POPSO)
N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO), N-tris (hydroxymethyl) Methyl-2-aminopropanesulfonic acid (TES), N- [Tris (hydroxymethyl) methyl] glycine (Tricine) and Trishydroxymethylaminomethane (Tris)
Etc.
Examples of most preferred buffering agents include, for example, trishydroxymethylaminomethane (Tris)
And piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) (POPSO).
The concentration of buffer used with these ASOx is ASOx even in the presence of protease.
Any amount may be used as long as the concentration is stable and does not affect the reaction of protease and ASOx. For example, the concentration is usually 1 mM to 1 M, preferably 5 mM to 500 mM.

As a protein denaturant of albumin and / or a compound having an S—S bond that can be used for accurately measuring glycated albumin using the present invention, the reactivity of BCP to albumin is G.
Anything can be used as long as A and NGA match.
Examples of preferred protein denaturing agents include, for example, urea, guanidine compounds, anionic surfactants such as sodium lauryl sulfate (SDS), polyoxyethylene alkylphenyl ether sulfate, polyoxyethylene alkyl ether sulfate, alkylbenzene. Examples thereof include sulfonates, and these can be used alone or in combination. In addition, the use concentration of these protein denaturing agents may be any concentration at which the reactivity of BCP to albumin is consistent with GA and NGA, for example, usually 0.01% to 10%, preferably 0.05%.
It may be used at a concentration of ˜5%.
Preferred compounds having an S—S bond include 6,6′-dithiodinicotinic acid (6,6′-dithiodin
icotinic acid), 3,3'-dithiodipropionic acid, 2,2'-dithiodisalicylic acid, 4,4'-dithiodimorpholine (4 , 4'-dithiod
imorpholine), 2,2'-dihydroxy-6,6'-dinaphthyl disulfide (2.2'-dihydroxy-6,6'-
dinaphthyl disulfide (DDD), 2,2'-dithiodipyridine (2-PDS), 4,4
'-Dithiodipyridine (4,4'-dithiopyridine; 4-PDS), 5,5'-dithiobis (2-nitrobenzoic acid)
(5,5'-dithiobis- (2-nitrobenzoic acid); DTNB), 2,2'-dithiobis (5-nitropyridine) (2
, 2'-dithiobis- (5-nitropyridine)) and the like.
In addition, the use concentration of the compound having an SS bond may be a concentration at which the reactivity of BCP to albumin is consistent with GA and NGA, for example, usually 1 μM to 10 mM, preferably 10 μM to 5 mM. Any other concentration may be used.

As a protease stabilizer that can be used in accurately measuring glycated protein using the present invention, any substance that suppresses a decrease in protease activity during reagent storage may be used. Any substance that suppresses a decrease in protease activity during storage in a liquid state is preferable.
Examples of preferred stabilizers include dimethyl sulfoxide, alcohol, water-soluble calcium salt, sodium chloride, quaternary ammonium salt or quaternary ammonium salt type cationic surfactant. Examples of alcohols include ethanol, propanol, ethylene glycol, glycerin and the like, and examples of quaternary ammonium salt quaternary ammonium salt type cationic surfactants include lauryl sulfate triethanolamine, lauryltrimethylammonium chloride, and the like. It is done.
These protease stabilizers may be used at any concentration as long as it is a substance that suppresses the decrease in protease activity during storage of the reagent. Particularly, the protease activity decreases during storage of the reagent in a liquid state. If it is the density | concentration which suppresses, it is preferable. As an example of a preferable concentration, for example, a concentration of usually 0.01% to 30%, preferably 0.1% to 20% may be used, and other concentrations may be used.

As an enzyme stabilizer that acts on at least a glycated amino acid that can be used in accurately measuring a glycated protein using the present invention, a substance that suppresses at least a decrease in the activity of an enzyme that acts on a glycated amino acid during storage of the reagent can be used. Any substance may be used as long as it is a substance that suppresses at least a decrease in the activity of an enzyme that acts on a glycated amino acid during storage of the reagent in a liquid state.
Examples of preferable stabilizers include sugar alcohol, sucrose, water-soluble magnesium salt, water-soluble calcium salt, ammonium sulfate, amino acid, and sarcosine. Examples of sugar alcohols include sorbitol, mannitol, trehalose, glycerin, etc., and as amino acids, all amino acids have a strong stabilizing effect, but more preferred are proline, glutamic acid, alanine, valine, glycine, lysine, etc. It is done.
In addition, the concentration of the stabilizer for the enzyme that acts on at least the glycated amino acid may be used at any concentration as long as it is a substance that suppresses at least a decrease in the activity of the enzyme that acts on the glycated amino acid during storage of the reagent. It is preferable to use a concentration that suppresses at least a decrease in the activity of the enzyme acting on the glycated amino acid during storage of the reagent in a liquid state. Examples of preferred concentrations include
For example, in the case of sugar alcohol, sucrose, amino acid, sarcosine, it may be used usually at a concentration of 0.01% to 30%, preferably 0.1% to 20%, and in the case of water-soluble magnesium salt, water-soluble calcium salt, ammonium sulfate, usually 1 mM to It may be used at a concentration of 1M, preferably 10 mM to 500 mM, and may be used at other concentrations.

The liquid composition in the composition for measuring glycated protein of the present invention is appropriately combined so that a proteolytic reagent containing a protease and a glycated amino acid measuring reagent for measuring the generated glycated amino acid or peptide can be used in the same reaction vessel. It ’s fine. In addition, these reagents can be provided as liquid products and liquid frozen products or freeze-dried products.
As a proteolytic reagent composition that can be used in the present invention, pH, buffer and protease concentration are determined so that the proteolytic reaction proceeds efficiently, and then a globulin component selective protease inhibitor, ASOx, and protease stabilizer May be appropriately prepared and added so as to have the above-mentioned effective concentration.
For example, when protease type XXIV (manufactured by Sigma) is used, the proteolytic activity is strong at a pH of around 7 to 10, and the pH of the reaction can be selected from 7 to 10. The buffer is a buffer having no 4- (2-hydroxyethyl) -1-piperazinyl group, for example, POPS having a buffering action at pH 7.2 to 8.5.
An O buffer solution can be used, and the concentration of POPSO may be 1 to 100 mM, preferably 10 to 500 mM.
The protease addition concentration is sufficient if it can sufficiently decompose the glycated protein in the test solution during the reaction time actually used, preferably 100 to 500,000 PU / ml, more preferably 500 to 100,000 PU / ml. More preferred is ml.
As a combination with a globulin component-selective protease inhibitor, ASOx, and a protease stabilizer, for example, as a globulin component-selective protease inhibitor, sulfate-3-[(cholamidopropyl) dimethylammonio] -1-propane, 0.01% to 20%, preferably 0.05%
~ 10%, pumpkin-derived ascorbate oxidase (manufactured by Toyobo), 0.1 U / ml to 100 U / ml,
Preferably 1 U / ml to 50 U / ml, dimethyl sulfoxide 0.01% as protease stabilizer
-30%, preferably 0.1% -20%, etc. can be used.

Regarding the reagent composition for measuring glycated amino acid that can be used in the present invention, the pH is selected so that the reaction proceeds efficiently in consideration of the optimum pH of the enzyme that acts on at least the glycated amino acid to be used, and then acts on the glycated amino acid. The amount of the enzyme is determined, and finally an enzyme stabilizer that acts on at least the glycated amino acid may be added.
For example, when using R-FOD or R-FOD-II (manufactured by Asahi Kasei Co., Ltd.), the region showing 50% or more of the maximum activity is as wide as pH 6.5 to 10, and the reaction pH can be selected from 6.5 to 10. . In addition, the enzyme addition concentration only needs to be a concentration at which glycated amino acids can be sufficiently detected in the reaction solution to be used.
-200 U / ml is preferred, and 1-50 U / ml is more preferred.
As a stabilizer for an enzyme that acts on at least a glycated amino acid, for example, glutamic acid can be used, and it may be used at a concentration of 0.01% to 30%, preferably 0.1% to 20%.

The first reagent that can be used in the present invention includes an enzyme that acts on at least a glycated amino acid, and a composition that contains a protease in the second reagent. For example, any conditions may be used as long as pH, salt concentration, etc. are set and conditions suitable for preservation of protease are set for the second reagent.
For example, when using R-FOD and protease type XXIV, pH 6
. Since the enzyme works well at 5 to 10 and 7 to 10, the pH of the first reagent may be 7 to 10 and a relatively high concentration of, for example, 20 to 1000 mM buffer may be selected. On the other hand, since this protease is stable at pH 7 or less, the pH of the second reagent is selected to be 7 or less, and a relatively low concentration of, for example, 1 to 50 mM buffer is selected from that of the first reagent. It is preferable to add a protease stabilizer of about 1 to 50% of oxide. In this case, if the mixing ratio of the first reagent and the second reagent is, for example, 4: 1 and the first reagent is increased, a higher concentration stabilizer, a pH far from the first reagent, and the like can be selected.

Further, for example, surfactants, salts, buffers, pH adjusters, preservatives and the like may be appropriately selected and added to the enzyme reaction composition for measuring the glycated protein according to the present invention.
As the surfactant, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, 0.01% to 10%, preferably 0.05% to 5%, such as polyvinyl alcohol,
Various metal salts such as lithium chloride, sodium chloride, potassium chloride, manganese chloride, cobalt chloride, zinc chloride, calcium chloride, etc. 1 mM to 5 M, preferably 10 mM to 1 M, various buffers
For example, Tris-HCl buffer, glycine-NaOH buffer, phosphate buffer, Good's buffer, etc.
mM to 2M, preferably 20 mM to 1M, and various preservatives such as 0.01 to 10%, preferably 0.05 to 1% of sodium azide may be added as appropriate.

As a method for measuring a glycated protein that can be used in the present invention, 0.001 to 0.5 ml of a test solution is added to the composition for measuring a glycated protein of the present invention and reacted at a temperature of 37 ° C. to perform a rate assay. Is changed coenzyme in a few minutes to several tens of minutes after a certain time after the start of the reaction, for example, 1 minute after 3 minutes and 4 minutes, or 5 minutes after 3 minutes and 8 minutes, The amount of dissolved oxygen, hydrogen peroxide or other products may be measured directly or indirectly by the above method. In the case of an endpoint assay, the changed coenzyme, dissolved oxygen, excess The amount of hydrogen oxide or other products may be measured similarly. In this case, the amount of glycated protein in the test solution can be determined by comparing with changes in absorbance or the like when measured using a glycated protein at a known concentration.

The detection of the enzyme action that acts on at least a glycated amino acid that can be used in the present invention is carried out by, for example, directly measuring the amount of change in the coenzyme when using dehydrogenase, Indirect measurement may be performed using an electron carrier such as methosulphate and a reducing coloring reagent such as nitrotetrazolium, WST-1, 8 (manufactured by Dojin Chemical Laboratories) and various tetrazolium salts. You may measure directly and indirectly by well-known methods other than.
For example, when oxidase is used, it is preferable to measure the amount of oxygen consumed or the amount of reaction products. For example, when R-FOD is used as a reaction product, hydrogen peroxide and glucosone are produced by the reaction, and both hydrogen peroxide and glucosone are directly obtained by known methods.
It can be measured indirectly.
The amount of hydrogen peroxide may be measured by, for example, coloration, luminescence, fluorescence, etc. by producing a dye using peroxidase or the like, or may be measured by an electrochemical method, using catalase or the like. Then, an aldehyde may be generated from the alcohol, and the amount of the aldehyde generated may be measured.

In the presence of peroxidase, the hydrogen peroxide coloring system produces dyes by oxidative condensation of couplers such as 4-AA or 3-methyl-2-benzothiazolinone hydrazone (MBTH) and chromogens such as phenol. Or a leuco-type reagent that directly oxidizes and colors in the presence of peroxidase.
As the chromogen of the Trinder type reagent, phenol derivatives, aniline derivatives, toluidine derivatives and the like can be used. As specific examples, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine ( TOOS), N, N bis (4-sulfopropyl) -3-methylaniline disodium (TODB) (manufactured by Doujin Chemical Laboratory Co., Ltd.) and the like.
Specific examples of leuco reagents include N- (carboxymethylaminocarbonyl) -4,4-
Examples thereof include bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67) (manufactured by Wako Pure Chemical Industries, Ltd.).
In the fluorescence method, a compound that emits fluorescence by oxidation, for example, homovanillic acid, 4-hydroxyphenylacetic acid, or the like can be used. In the chemiluminescence method, luminol, lucigenin, isoluminol, or the like can be used as a catalyst.

In addition, when measuring hydrogen peroxide using an electrode, the electrode is not particularly limited as long as it is a material that can exchange electrons with hydrogen peroxide, and examples thereof include platinum, gold, and silver. As an electrode measurement method, a known method such as amperometry, potentiometry, coulometry, etc. can be used. Further, an oxidation or reduction current obtained by interposing an electron carrier in the reaction between the oxidase or substrate and the electrode Alternatively, the amount of electricity may be measured.
Any substance having an electron transfer function can be used as the electron carrier, and examples thereof include substances such as ferrocene derivatives and quinone derivatives. Alternatively, the oxidation or reduction current obtained by interposing an electron carrier between hydrogen peroxide generated by the oxidase reaction and the electrode, or the amount of electricity thereof may be measured.

When the glycated protein is glycated albumin and the glycated albumin ratio is accurately measured, the albumin measuring reagent that can be used in the present invention includes a protein denaturant and / or a compound having an S—S bond and bromocresol purple. Any composition may be used as long as it is prepared as a composition to be contained and does not cause a difference in measurement between GA and NGA.
For example, when sodium lauryl sulfate and 5,5′-dithiobis (2-nitrobenzoic acid) are used as a protein denaturant and / or a compound having an S—S bond, the concentration is low so as not to affect the color development of BCP. Buffer solution, for example, 1-20 mM, sodium lauryl sulfate 0.01% -10%
The concentration may be 0.05% to 5%, and 5,5′-dithiobis (2-nitrobenzoic acid) 1 μM to 10 mM, preferably 10 μM to 5 mM. BCP is neutral and violently colored.
It is good to use in .5-7.5.

As a method for measuring albumin that can be used in the present invention, 0.001 to 0.5 ml of a test solution is added to the composition for albumin measurement of the present invention, and reacted at a temperature of 37 ° C. What is necessary is just to measure the quantity of the pigment | dye after a fixed time. The detection of albumin-BCP color development is preferably performed at around 550 nm to 630 nm because the absorption maximum is around 600 nm. In this case, the amount of albumin in the test solution can be determined by comparing the absorbance with a known concentration of albumin and a blank of water.

As the test liquid to be measured in the present invention, any test liquid containing at least a glycated protein may be used, but preferred test liquids include blood components such as serum, plasma, blood cells, whole blood Examples include blood. Separated erythrocytes can also be used as a preferred test solution because the globulin component may be mixed depending on the separation conditions and affect the measured value.
Examples of the glycated protein to be measured in the glycated protein measurement composition and measurement method of the present invention include GA or GHb, but the glycated protein to be measured is not limited to these, and any The glycated protein may be measured.
Next, examples of the present invention will be described in detail, but the present invention is not limited thereto.

In order to screen for proteases that do not act on the globulin component, glycated amino acids or glycated peptides produced by reacting the protease with albumin, globulin component and hemoglobin were measured with R-FOD (Asahi Kasei Co., Ltd.).
<Substrate solution>
1; HSA substrate solution; Albumin Human; Essencially Globulin Free; 25mg / ml, GA% = 31.9%
, Fructosamine (FRA) value = 256 μmol / L [manufactured by Sigma; albumin concentration in substrate solution is measured with albumin measurement kit (albumin II-HA test Wako; manufactured by Wako Pure Chemical Industries, Ltd.), GA% is glycated albumin meter (GAA-2000; manufactured by Kyoto Daiichi Kagaku).
2; G-II, III substrate solution, FRA value 48μmol / L [Globulins Human Cohn Fraction II, III;
16.9mg / ml (Sigma)
3; G-IV substrate solution, FRA value 26 μmol / L [Globulins Human Cohn Fraction IV; 6 mg / ml (manufactured by Sigma)]
4; GI substrate solution, FRA value 77μmol / L [globenin I; glovenin-I; immunoglobulin preparation (manufactured by Takeda)]
5; Hb substrate solution; Hemoglobin Human; 55 mg / ml, glycated hemoglobin rate; HbA1c = 4.5% Measured with ]
The fructosamine value of the substrate solution was measured with a fructosamine measurement kit (Autowaco Fructosamine, manufactured by Wako Pure Chemical Industries, Ltd.).
<Protease reaction sample preparation>
Substrate solution other than Hb 200 μl, protease solution 100 mg / ml (if this concentration cannot be made, the highest possible concentration, and the solution is the same as it is) 40 μl, and 1 M Tris buffer (p
H8) 10 μl was mixed well and reacted at 37 ° C. for 30 minutes, filtered through a 10,000 membrane (Ultra Free MC; manufactured by Millipore), and the filtrate was used as a protease reaction sample. Moreover, the same operation was performed using distilled water instead of the substrate to obtain a blank sample.
On the other hand, in the case of the Hb substrate solution, 150 μl of the substrate solution, 200 mg / ml of the protease solution (if this concentration cannot be made, the highest possible concentration, or the solution-like one as it is) 60 μl and 1M
5 μl of Tris buffer (pH 8) was mixed well, reacted at 37 ° C. for 60 minutes, filtered through a 10,000 membrane (Ultra Free MC; manufactured by Millipore), and the filtrate was used as a protease reaction sample. Moreover, the same operation was performed using distilled water instead of the substrate to obtain a blank sample.

<Measurement of glycated amino acid and glycated peptide in protease reaction sample>
<Reaction solution composition>
50 mM Tris buffer pH 8.0
0.02% 4-AA (Wako Pure Chemical Industries, Ltd.)
0.02% N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine
(TOOS) (Doujin Chemical Laboratory Co., Ltd.)
2U / ml R-FOD (Asahi Kasei)
5U / ml POD (Sigma)
<Reaction procedure>
Dispense 300 μl of the above glycated amino acid measurement reaction solution into a cell and incubate at 37 ° C. for 3 minutes.
Measure 5nm (A0). Subsequently, 30 μl of protease reaction sample is added, incubated at 37 ° C. for 5 minutes, and 555 nm is measured (A1). In addition, a blank sample is used in place of the protease reaction sample, and the same operation is performed to measure A0 blank and A1 blank. When the action of protease on glycated protein is expressed by the change in absorbance, the following formula is obtained.
ΔA = (A1-A0)-(A1 blank-A0 blank)
Table 1 shows the action (ΔA) of typical proteases at pH 8.0 on albumin, globulin, and hemoglobin.

Table 1 shows that the effect on glycated protein in G-IV substrate solution was small or 0 for all proteases, and the measured values of G-II and -III substrate solutions were almost the same as those of GI substrate solutions. Shows only the results of the GI substrate solution for the globulin component. As can be seen from Table 1, proteases derived from the genus Aspergillus and protease type XIV also act well on glycated globulins in the globulin component.
However, endo-type and exo-type proteases that act on GA in albumin and GHb in hemoglobin both act on glycated globulin in the globulin component. Therefore, when measuring GA in serum or plasma or whole blood and GHb in blood cells, it was considered that the influence of globulin components could not be avoided only by selecting protease.

<Screening for globulin component selective protease inhibitors>
Using protease type XXIV (manufactured by Sigma) showing high action on the HSA substrate solution, H
Based on the SA substrate solution, substances that reduce the protease action on the various globulin substrate solutions were screened.
<Reaction solution composition>
R-1 Proteolytic reagent
150 mM Tricine buffer (Wako Pure Chemical Industries, Ltd.) pH 8.5
2500 U / ml protease type XXIV (manufactured by Sigma + globulin selective protease inhibitor (deoxycholic acid, deoxycholic acid amide, cholic acid amide, quaternary ammonium salt or quaternary ammonium salt type cationic surfactant; 1%, concanavalin A; 0.
(21mg / ml, betaine; 0.1%, octyl glucoside; 1%; manufactured by Doujin Chemical Laboratory)
R-2 Reagent for measuring glycated amino acid
150 mM Tricine buffer (Wako Pure Chemical Industries, Ltd.) pH 8.5
0.12% 4-AA (Wako Pure Chemical Industries, Ltd.)
0.08% TOOS (manufactured by Doujin Chemical Laboratory)
24U / ml R-FOD (Asahi Kasei Kogyo)
20U / ml POD (Sigma)
R-1 Deoxycholic acid amide in the proteolytic reagent is bisgluconamidopropyl deoxycholamide, and cholic acid amide is sulfuric acid-3-[(cholamidopropyl) dimethylammonio] -1-propane, sulfuric acid -3-[(Coleamidopropyl) dimethylammonio] -2-hydroxy-1-propane or bisgluconamidopropylcoramide, quaternary ammonium salts include benzyltriethylammonium chloride, benzyltri-n-butylammonium chloride, As the quaternary ammonium salt type cationic surfactant, lauryltrimethylammonium chloride and lauryldimethylamine oxide were used.
<Substrate solution>
1; HSA substrate solution; Albumin Human; 40 mg / ml, GA% = 10.5% [manufactured by Wako Pure Chemical Industries, Ltd. GA% was measured with a glycated albumin meter (GAA-2000; manufactured by Kyoto Daiichi Kagaku). ]
2; γ globulin added substrate solution; γ globulin s Human
(Sigma) Fructosamine value 34 μM] 17.0 mg / ml was added.
<Reaction procedure>
R-1 incubated at 37 ° C; 240 μl substrate solution (HSA substrate solution, GI substrate solution) 8 μl
The reaction was started at 37 ° C., and exactly 2 minutes later, R-2; 80 μl was added. 546 before and after R-2 addition
The absorbance at nm was measured, and the difference was defined as a change in absorbance. In addition, the same operation was performed using distilled water instead of the substrate to obtain a blank sample, and a reaction solution to which a globulin-selective protease inhibitor was not added was used as a control.
The change in absorbance obtained from the HSA substrate solution minus ΔA (H
SA) and ΔA (+ γ globulin) calculated by subtracting the absorbance change of the blank sample from the absorbance change obtained from the γ globulin added substrate solution,
Effect of gamma globulin addition
= (ΔA (+ γ globulin) -ΔA (HSA)) / ΔA (HSA) × 100 (%)
Comparison was made in the presence and absence (control sample) of various candidate substances. The results are shown in Table 2.

As can be seen from Table 2, deoxycholic acid, deoxycholic acid amide, cholic acid amide,
Quaternary ammonium salt or quaternary ammonium salt type cationic surfactant, concanavalin A, octyl glucoside and betaine have been confirmed to inhibit the protease action on globulin, and these globulin component selective protease inhibitors and proteases It became clear that proteins other than globulins can be fragmented by using.
Furthermore, the same measurement was performed using an Hb substrate solution instead of the HSA substrate solution. However, when the Hb substrate solution was used, after completion of the R-1 reaction, the protein was deproteinized with trichloroacetic acid, neutralized and then R-2 was added for evaluation. Proteolytic action on globulin in deoxycholic acid, deoxycholic acid amide, cholic acid amide, quaternary ammonium salt or quaternary ammonium salt type cationic surfactant, concanavalin A and betaine even when Hb substrate solution is used The effect which inhibits was confirmed.

<Sulfuric acid-3-[(Coleamidopropyl) dimethylammonio] -1-propane globulin component selective protease inhibitory effect>
Using various proteases, sulfate-3-[(cholamidopropyl) dimethylammonio] -1-
Protease inhibitory effect of propane was confirmed.
R-1 Proteolytic reagent
150 mM Tricine buffer (Wako Pure Chemical Industries, Ltd.) pH 8.5
2500U / ml protease *
1% Sulfate-3-[(Choleamidopropyl) dimethylammonio] -1-propane * Protease is Orientase 22BF (Hankyu Bioindustry), Prote
Aase type VIII, protease type XIV, protease type XXVII (or above)
Sigma) was used.
R-2 Saccharified Amino Acid Measuring Reagent Same as Example 2 <Substrate Solution>
Same as Example 2.
<Reaction procedure>
The effect of adding gamma globulin in the same procedure as in Example 2 was compared in the presence and absence (control sample) of sulfuric acid-3-[(cholamidopropyl) dimethylammonio] -1-propane.
The results are shown in Table 3. In the column for determination, “◯” was given when the effect of the addition of γ globulin decreased significantly.

As can be seen from Table 3, orientase 22BF, protease type VIII, protease type XIV, and protease type XXVII are all proteases to gamma globulin substrates in the presence of sulfate-3-[(cholamidopropyl) dimethylammonio] -1-propane. The action is reduced, whereas HS
The effect on the A substrate was retained. From this, it became clear that the globulin component selective protease inhibitor in the present invention is effective regardless of the type of protease.
Further, when measuring GHb in the same manner, the influence of the globulin component could be avoided by using the present invention.

<Dilution linearity of glycated albumin>
R-1 Proteolytic reagent
150 mM Tricine buffer (Wako Pure Chemical Industries, Ltd.) pH 8.5
2500U / ml Protease type XXVII (Sigma)
1% Sulfuric acid-3-[(Coleamidopropyl) dimethylammonio] -2-hydroxy-1-
Propane (Sigma)
R-2 Saccharified Amino Acid Measuring Reagent Same as Example 2 <Substrate Solution>
1: HSA substrate solution; same as Example 1. However, the concentration was 4.0 g / dl.
2: γ globulin substrate solution; same as in Example 2.
3: Globulin IV substrate solution; same as Example 1.
<Operation>
HSA substrate solution (4g / dl), γ globulin (γG) and globulin IV (GIV) substrate solution 1.7g / dl)
Samples of 0.0, 0.5, 1.0, 1.5, and 2.0 times the concentration were prepared, and dilution linearity was confirmed. The operation is the same as in Example 3. However, the HSA 1.0-fold concentration was measured 10 times and the CV value was calculated. The result is shown in FIG.

As can be seen from FIG. 1, no change in absorbance was obtained even when the concentrations of both the gamma globulin and globulin IV substrate solutions were changed. On the other hand, the HSA substrate solution has good linearity depending on the concentration,
It is clear that glycated albumin is measured substantially unaffected by the globulin component. In addition, it has been confirmed that the reproducibility of the CSA value is 0.9% at an HSA of 1.0 times, and the glycated albumin is selectively and sensitively and reproducible in a reaction time of 10 minutes using the measurement system of the present invention It became clear that it was measured.

<Dilution linearity of glycated hemoglobin>
R-1 Proteolytic reagent
77 mM Tris buffer, pH 8.0
2500U / ml protease type XIV (manufactured by Sigma)
1% Sulfuric acid-3-[(cholamidopropyl) dimethylammonio] -2-hydroxy
Si-1-propane (Sigma)
R-2 Glycated amino acid measurement reagent Same as Example 2.
<Substrate solution>
The same Hb substrate solution as in Example 1 and the same γ globulin and globulin IV substrate solution as in Example 4 were used.
<Operation>
0.0, 0.5, 1. of Hb substrate solution (4 g / dl), gamma globulin and globulin IV substrate solution (1.7 g / dl)
Samples with concentrations of 0, 1.5, and 2.0 were prepared to confirm dilution linearity. The operation is the same as in Example 1. Where H
b1.0 concentration was measured 10 times and CV value was calculated. The result is shown in FIG.

As can be seen from FIG. 2, no change in absorbance was obtained even when the concentrations of both the gamma globulin and globulin IV substrate solutions were changed. On the other hand, the Hb substrate solution has good linearity depending on the concentration, and it is clear that glycated hemoglobin is measured without being substantially affected by the globulin component. In addition, a good reproducibility of CV value = 2.0% was confirmed at a Hb 1.0-fold concentration, and glycated hemoglobin was selectively and highly sensitive and reproducible in a reaction time of 10 minutes using the measurement system of the present invention. It became clear that it was measured.

<Linearity of glycated albumin>
R-1 Proteolytic reagent Same as Example 4.
R-2 Glycated amino acid measurement reagent Same as Example 4.
<Substrate solution>
Serum A) * Diabetes patient serum GA% = 32.9%; albumin concentration 4.3 g / dl
Serum B) * Serum in normal subjects GA% = 16.4%; albumin concentration 4.1 g / dl
* The above serum A) and B) were mixed at a ratio of 10: 0, 8: 2, 6: 4, 4: 6, 2: 8, 0:10 to prepare a sample.
<Operation> Same as Example 3.
The results are shown in FIG.

As can be seen from FIG. 3, good linearity was obtained even in samples with different glycated albumin ratios with a constant albumin concentration. Therefore, it was found that glycated albumin in actual serum and plasma can be quantitatively detected by the method for measuring glycated protein of the present invention. In addition, since the same linearity was confirmed using a hemoglobin substrate solution prepared by lysing red blood cells instead of serum, it was also confirmed that glycated hemoglobin could be quantitatively detected by the method for measuring glycated protein of the present invention. .

<Correlation between glycated albumin HPLC method and enzyme method (present invention)>
R-1 Proteolytic reagent Same as Example 4.
R-2 Glycated amino acid measurement reagent Same as Example 4.
<Substrate solution> Diabetes patient serum 14 samples
Serum of 25 healthy subjects <Operation> The procedure is the same as in Example 2.
The correlation between the enzyme method based on the present invention and a known HPLC method was confirmed using 14 serum samples of diabetic patients. In the HPLC method, the glycated albumin ratio was measured with a glycated albumin meter (GAA-2000; manufactured by ARKRAY). The absorbance change obtained from the measuring method according to the present invention is the glycated albumin rate,
The correlation coefficient r = 0.991 showed a very good correlation, and it became clear that the measurement method based on the present invention accurately measured glycated albumin.

<Effect of buffer type on stabilization of ascorbate oxidase>
<Reaction solution composition>
150mM various buffers pH8.0
2500U / ml Protease type XXIV (Sigma) or Pronase (Sigma)
Made)
10 U / ml ascorbate oxidase (ASO-311; manufactured by Toyobo) or heat stable
Type ascorbate oxidase (ASO-312; manufactured by Toyobo)
R-1 3- [4- (2-hydroxyethyl) -1-piperazinyl] as a buffer in proteolytic reagents
Propanesulfonic acid (EPPS), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] Propanesulfonic acid (HEPPSO), trishydroxymethylaminomethane (Tris), piperazine-1,4
-Bis (2-hydroxy-3-propanesulfonic acid) (POPSO) (manufactured by Doujin Chemical Laboratory Co., Ltd.) and phosphoric acid (manufactured by Wako Pure Chemical Industries, Ltd.) were used.
<Operation procedure>
The said reaction liquid was created using various buffering agents, the one part was taken, and the ascorbate oxidase activity in a reagent was measured and it was set as control. As the method for measuring the activity, the aforementioned << method for measuring the activity of ascorbate oxidase (ASOx) >> was used. The remaining reaction solution was stored at room temperature for 2 days, and the activity was measured in the same manner. The ratio of the activity after storage at room temperature for 2 days to the activity of the control was calculated, and the stability of ascorbate oxidase was compared. The results are shown in Table 4.

As can be seen from Table 4, 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS) having 4- (2-hydroxyethyl) -1-piperazinyl group, 2- [4- ( When 2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) and 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (HEPPSO) are used as buffers Than 4
-(2-hydroxyethyl) -1-piperazinyl group-free trishydroxymethylaminomethane (Tris), piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) (POPSO) and phosphoric acid buffer It was clear that ascorbate oxidase existed more stably in the presence of protease.
It is also clear that similar effects have been confirmed regardless of the type of ascorbate oxidase or protease.

<Effects of buffer type on stabilization of ascorbate oxidase in glycated protein measurement composition>
<Reaction solution composition>
R-1 Proteolytic reagent
150mM various buffers pH8.0
2500U / ml protease type XXIV (manufactured by Sigma)
2.0 mM 4-aminoantipyrine (Wako Pure Chemical Industries, Ltd.)
10U / ml ascorbate oxidase (Toyobo)
R-2 Reagent for measuring glycated amino acid
150 mM HEPES buffer (Wako Pure Chemical Industries, Ltd.) pH 7.5
6.0mM TOOS (Doujin Chemical Laboratory)
24U / ml R-FOD (Asahi Kasei)
20U / ml POD (Sigma)
EPPS, HEPES, HEPPSO, Tris and POPSO were used as buffers in the R-1 proteolytic reagent.
<Control substrate solution, substrate solution with ascorbic acid added>
1 volume of ascorbic acid (manufactured by Kokusan Chemical Co., Ltd.) 1 g / dl was added to 9 volumes of human pooled serum to obtain an ascorbic acid-added substrate solution. A control substrate solution was prepared by adding distilled water instead of ascorbic acid.
<Reaction procedure>
8 μl of control substrate solution or ascorbic acid-added substrate solution was added to 240 μl of R-1 incubated at 37 ° C., the reaction was started at 37 ° C., and R-2; 80 μl was added exactly 5 minutes later. Absorbance at 555 nm was measured before the addition of R-2 and 5 minutes after the addition. In addition, the measurement using distilled water instead of the substrate solution was used as a blank, and ΔA0 was calculated by subtracting the absorbance change of the blank sample from the absorbance change obtained from the control substrate solution and the substrate solution containing ascorbic acid. On the other hand, the same reaction solution R-1 was stored at room temperature for 24 hours, the same measurement was performed, and ΔA24 was calculated. ΔA obtained from an ascorbic acid-added substrate solution with the change in absorbance obtained from the control substrate solution as 100
The ratio of 0 and ΔA24 was calculated. The results are shown in FIG.

Ascorbic acid has a large negative effect on the measurement system, and if the concentration is 100 mg / dl, the signal of glycated protein cannot be observed unless an elimination reaction is performed. As can be seen from FIG. 4, the ability to treat ascorbic acid does not change even in the system for measuring glycated protein in Tris and POPSO without 4- (2-hydroxyethyl) -1-piperazinyl group after storage at room temperature for 24 hours, while EPPS , HEPES and HEP
In the buffer system with 4- (2-hydroxyethyl) -1-piperazinyl group using PSO, ascorbic acid treatment ability was hardly observed after storage at room temperature for 24 hours. From the above results, in the glycated protein measurement reagent in which protease and ascorbate oxidase coexist, 4- (2-hydroxyethyl) -1-piperazinyl group is used more than 4- (2-hydroxyethyl) -1-piperazinyl group. It was clear that ascorbate oxidase was present more stably when used in a buffer without an ethyl) -1-piperazinyl group.
It is also apparent from the results that the present invention is useful for the measurement of glycated albumin, fructosamine and glycated hemoglobin.

<Differences in the action of bromocresol purple on glycoalbumin and non-glycoalbumin, and the effects of protein denaturants and / or compounds having an SS bond>
<Reaction solution composition>
R-1 pretreatment reagent
10 mM Tris-HCl buffer pH 8.0 + protein denaturant at each concentration and / or SS
A compound having a bond, distilled water was added as a control.
R-2 Albumin coloring reagent
200mM succinate buffer (Wako Pure Chemical Industries, Ltd.) pH5.5
0.15mM Bromocresol Purple (Wako Pure Chemical Industries, Ltd.)
0.3% Tx-100 (Wako Pure Chemical Industries)
The following 1) to 9) were used as a protein denaturant and / or a compound having an S—S bond in the R-1 pretreatment reagent.
1) 6,6'-dithiodinicotinic acid (100mM)
2) 3,3'-dithiodipropionic acid (100mM)
3) 2,2'-dithiodisalicylic acid (2,2'-dithiodibenzoicacid; 100mM)
4) 4,4'-dithiodimorpholine (100mM)
5) DTNB (50mM)
6) DDD (33mM)
7) 2-PDS (25mM)
8) 4-PDS (50mM)
9) SDS (0.3%)
1) to 5) Wako Pure Chemical Industries, 6) to 9) Doujin Chemical Laboratory Co., Ltd. <Sample>
Glycoalbumin, non-glycoalbumin, healthy subject serum and patient serum were used as samples, and distilled water was used as a blank. For glycoalbumin and non-glycoalbumin, albumin was purified from human serum by a known method and purified using a boric acid-immobilized resin.
<Reaction procedure>
Pretreatment reagent incubated at 37 ° C .; 2 μl of sample was added to 160 μl, the reaction was started at 37 ° C., and exactly 5 minutes later, albumin coloring reagent; 160 μl was added. Absorbance at 600 nm was measured before and 5 minutes after addition of the albumin coloring reagent. In addition, a calibration curve was prepared from a sample having a known concentration of distilled water and albumin instead of the sample, and an albumin measurement value was obtained. Further, an immunization method using a latex reagent (LX reagent, manufactured by Eiken Co., Ltd., Alb-II) was separately measured and used as a control. The results are shown in Table 5.

As can be seen from Table 5, the BCP method without pretreatment unexpectedly shows a low value in NGA, and the difference between the immune method and the BCP method is similar to that in healthy subjects with a lot of NGA. It was small. In addition, the pretreatment with a protein denaturant and / or a compound having an S—S bond significantly reduced the deviation from the immunization method. Among them, the effects of 2,2′-dithiosalicylic acid, 4,4′-dithiodimorpholine, DDD, 2-PDS, 4-PDS, DTNB and sodium lauryl sulfate were remarkable. From this, as an albumin reagent used when measuring the ratio of glycated albumin,
By using a method in which albumin is measured by pretreatment with a protein denaturant and / or a compound having an S—S bond, and subsequently or simultaneously with BCP, the phenomenon of giving a negative error by NGA is avoided. It became clear that the ratio of glycated albumin could be measured accurately.

<Stabilization of protease>
<Reaction solution composition>
R-1 Proteolytic reagent
150 mM Tris-HCl buffer pH 8.5
5000PU / ml protease type XXIV (manufactured by Sigma)
8 mM 4-aminoantipyrine (manufactured by Doujin Chemical Laboratory)
15U / ml peroxidase
1.0% Sulfuric acid-3-[(Choleamidopropyl) dimethylammonio] -2-hydroxy
Si-1-propane (Sigma) + protease stabilizer at various concentrations, control
As distilled water was added.
R-2 Reagent for measuring glycated amino acid
150 mM Tris-HCl buffer pH 8.5
24U / ml R-FOD-II (Asahi Kasei)
12mM TOOS (manufactured by Doujin Chemical Laboratory)
The following 1) to 7) were used as protease stabilizers in the pretreatment reagent.
1) 0.5mM magnesium chloride
2) 10mM calcium chloride
3) 100 mM sodium chloride
4) 0.1% ethylene glycol (EtGly)
5) 10% dimethyl sulfoxide (DMSO)
6) 1% ethanol (EtOH)
7) 0.1% Triananolamine lauryl sulfate (TEALS)
1)-7) Wako Pure Chemical Industries <Sample>
5g / dl HSA (manufactured by Sigma; LOT38H7601)
<Reaction procedure>
Proteolysis reagent incubated at 37 ° C .; 8 μl of sample was added to 240 μl, the reaction was started at 37 ° C., and glycated amino acid measurement reagent; 80 μl was added exactly 5 minutes later. Absorbance at 546 nm was measured before the addition of the glycated amino acid measurement reagent and 5 minutes after the addition. In addition, the measurement using distilled water instead of the substrate solution was used as a blank, and ΔA0 was calculated by subtracting the absorbance change of the blank sample from the absorbance change obtained from the substrate solution. On the other hand, the same reaction solution proteolytic reagent was stored at 37 ° C. for 24 hours, and the same measurement was performed to calculate ΔA24. 100% ΔA0 without stabilizer and without storage
The relative sensitivity with and without the stabilizer was calculated. The results are shown in FIG.

As can be seen from FIG. 5, in the absence of stabilizer, the relative sensitivity was reduced to 60%, and the stabilizing effect of the proteolytic reagent was observed. On the other hand, when a stabilizer was added, a stabilizing effect was observed for calcium chloride, sodium chloride, DMSO, EtOH, and TEALS, and among them, performance degradation was hardly observed for calcium chloride and DMSO. Furthermore, as a result of continuing the stability test for DMSO and calcium chloride, it was revealed that even when stored at 37 ° C. for 4 weeks, a decrease in performance was not observed, and the storage stability was 1 year or longer when stored in a liquid state and refrigerated.

<Stabilization of enzymes that act on at least glycated amino acids>
<Reaction solution composition>
R-1 Proteolytic reagent
150 mM Tris-HCl buffer pH 8.5
8 mM 4-aminoantipyrine (manufactured by Doujin Chemical Laboratory)
15U / ml peroxidase R-2 glycated amino acid reagent
150 mM Tris-HCl buffer pH 8.5
24U / ml R-FOD-II (Asahi Kasei)
12 mM TODB (manufactured by Dojin Chemical Laboratories) + protease stabilizer at various concentrations, control
As distilled water was added.
The following 1) to 15) were used as qualifiers for enzymes acting on at least glycated amino acids in the glycated amino acid measurement reagent.
1) 5% mannitol
2) 5% sorbitol
3) 5% sucrose
4) 5% trehalose
5) 0.5mM calcium chloride
6) 0.5mM magnesium chloride
7) 3% L-glutamic acid (Glu)
8) 3% L-glutamine (Gln)
9) 3% L-proline (Pro)
10) 3% L-alanine (Ala)
11) 3% L-Valine (Val)
12) 3% Glycine (Gly)
13) 3% L-Lysine (Lys)
14) 3% sarcosine
15) 100 mM ammonium sulfate
1) -14) Wako Pure Chemicals <Sample>
0.5mM FZL
<Reaction procedure>
Proteolysis reagent incubated at 37 ° C .; 8 μl of sample was added to 240 μl, the reaction was started at 37 ° C., and glycated amino acid measurement reagent; 80 μl was added exactly 5 minutes later. Absorbance at 546 nm was measured before the addition of the glycated amino acid measurement reagent and 5 minutes after the addition. In addition, the measurement using distilled water instead of the substrate solution was used as a blank, and ΔA0 was calculated by subtracting the absorbance change of the blank sample from the absorbance change obtained from the substrate solution. On the other hand, the same reaction solution glycated amino acid measurement reagent was stored at 37 ° C. for 2 days, and the same measurement was performed to calculate ΔA24. ΔA0 without stabilizer and without storage
The relative sensitivity was calculated with and without a stabilizer. The results are shown in FIG.

As can be seen from FIG. 6, in the absence of a stabilizer, the relative sensitivity decreased to 30%, and the stabilizing effect of the glycated amino acid measuring reagent was observed. On the other hand, when a stabilizer is added, the stabilizing effect is mannitol, sorbitol, sucrose, trehalose, calcium chloride, magnesium chloride, L-glutamic acid, L-glutamine, L-proline, L-alanine, L-valine, glycine, L-lysine, sarcosine, and ammonium sulfate were observed, and sugar alcohol, amino acid, and sarcosine were confirmed to have particularly strong stabilizing effects. Furthermore, L-alanine, glycine and sarcosine, as a result of continuing the stability test, almost no degradation in performance was observed even after storage at 37 ° C for 4 weeks,
It has become clear that it has a storage stability of over 1 year in refrigeration.

<Generation of mutant FOD gene-containing DNA fragment library>
Synthesis of oligonucleotides having a sequence of 1 to 30 of the base sequence of SEQ ID NO: 5 and oligonucleotides having a sequence of 1 to 30 of the base sequence of SEQ ID NO: 6 (BEX
And DNA encoding the FOD protein from Fusarium oxysporum IFO-9972
Was used as a template, PCR was performed using Taq polymerase kit (Takara Shuzo) according to the attached manual, and the FOD structural gene was amplified. At this time, 2 in the reaction solution corresponding to a final concentration of 0.5 mM.
Add valent manganese ion, base is dATP: 0.51 mM, dCTP: 0.20 mM, dGTP: 1.15 mM, dTTP: 3.76
The reaction was carried out with the concentration of mM and concentration unevenly, and the efficiency of mutagenesis was increased.

<Creation of mutant FOD recombinant library>
The FOD gene-containing DNA fragment amplified in Example 13 was digested with restriction enzymes NcoI and EcoRI, incorporated into plasmid oTV119N (Takara Shuzo) treated with the same restriction enzymes, and introduced into Escherichia coli JM109 strain (Toyobo Co., Ltd.). Then, it was cultured overnight at 37 ° C. on a LB agar plate medium (DIFCO) containing 100 μg / ml ampicillin to form transformant colonies.

<Screening of lysine-specific mutation FOD>
The library colonies prepared in Example 14 were treated with two 100 μg / ml ampicillin and 1 mM I.
Replicate to LB agar plate medium containing PTG (Wako Pure Chemical Industries) and 5 U / ml on each medium.
Peroxidase (Asahi Kasei), 0.02% orthodianisidine (Wako Pure Chemical Industries), 2.
0 mM glycated valine or glycated lysine (Hashiba, H. (1976) J. Agric. Food C
LB. agar (0.3%) medium containing hem., 24, 70)) and cultured for 8 hours at 37 ° C to develop color of colonies due to oxygen radicals generated by oxidation of glycated amino acids by FOD and dianisidine Observed. As a result, colonies that developed a dark purple color with glycated lysine and not colored with glycated valine were screened to obtain 164 corresponding colonies.

<Preparation of mutant FOD candidate cell extract>
The mutant colony 164 strain obtained in Example 15 was cultured at 37 ° C. for 16 hours in 1.5 ml of 3.7% BHI (DIFCO) liquid medium containing 50 μg / ml ampicillin and 1 mM IPTG, 1 ml of which was centrifuged. The cells are collected by separation (15,000G, 1 minute, 4 ° C), 200 μl of 10 mM Tris-HCl buffer (pH 8.0) is added, the cells are crushed using an ultrasonic crusher, and then centrifuged (14,000 G, 5 minutes, 4 ° C.), and the supernatant was obtained as a cell extract.

<Substrate specificity test of mutant FOD>
For the cell extract prepared in Example 16, the glycated amino acid substrate specificity of the contained FOD mutant recombinant was measured by the aforementioned FOD enzyme activity measurement method. As a result, we confirmed two mutants in which the activity to glycated valine is less than 1/1000 of the activity of glycated lysine in the candidate strain,
The target mutant was used.

<Extraction of recombinant plasmid>
The mutant selected in Example 17 was inoculated into 1.5 ml of LB liquid medium containing 50 μg / ml ampicillin 3
After shaking culture at 7 ° C. for 16 hours, the plasmid was extracted according to a conventional method. Pcm
They were named FOD1 and pcmFOD2.

<Determination of mutant FOD gene base sequence>
The nucleotide sequence of the mutant FOD gene obtained in Example 18 was determined by the dideoxy method. As a result, the two mutants have the same structure, A in the nucleotide sequence 1115 of SEQ ID NO: 1 is converted to G, and the amino acid sequence of the encoded recombinant mutant FOD is the amino acid sequence of SEQ ID NO: 1 It was confirmed that lysine # 372 was converted to arginine.

<Verification of substrate specificity of various mutants>
For the amino acid mutation site confirmed in Example 19, the site-specific mutation method of Kunkel et al. Was performed in order to observe the effect of substitution with another amino acid. Of the nucleotide sequence described in SEQ ID NO: 7
Outsourced synthesis of oligonucleotides with sequences from 1 to 27 (BEX), and
Using Mutan-K kit (Takara Shuzo), site-specific mutation was performed according to the attached manual. The obtained mutant gene is again incorporated into the expression plasmid pTV119N, introduced into an E. coli host, and introduced into a 3.7% BHI liquid medium containing 50 μg / ml ampicillin and 1 mM IPTG at 30 ° C., 16 ° C.
By culturing for a while, mutant FOD protein was produced. The substrate specificity of the plurality of mutants obtained as described above was measured in the same manner as in Examples 16 and 17, and in addition to arginine, tryptophan, methionine, threonine, valine, alanine, serine, cysteine, and glycine. It was confirmed that the converted mutant had the same glycated lysine-specific substrate specificity as the mutant converted to arginine. The results are shown in Table 6.

In addition, below the limit in the table below the measurement limit, ND indicates no measurement data. As a result, it was confirmed that the glycated lysine reactivity of FOD can be lowered relative to the glycated valine reactivity by converting the 372nd lysine of the amino acid sequence of SEQ ID NO: 1, especially tryptophan, methionine. The mutant to valine was found to have high glycated lysine specificity and good enzymatic properties. The mutant for tryptophan is FOD-W, the expression plasmid for producing FOD-W is pcmFOD3, the mutant for methionine is FOD-M, the expression plasmid for producing FOD-M is pcmFOD4, and the mutant for valine is FOD. PcmFOD5 is an expression plasmid that produces -V and FOD-V
Named. FIG. 7 shows the common structure of each plasmid.

<Erase of ε-fructosyl-L-lysine (ZFL) in the test solution, then fructosyl-L-valine (
FV)>
Reaction solution 1
50 mM Tris-HCl buffer (pH 7.5)
10U / ml FOD-V
5 U / ml catalase reaction solution 2
50 mM Tris-HCl buffer (pH 7.5)
10U / ml FOD
20U / ml peroxidase
0.05% sodium azide
0.04% 4-aminoantipyrine
0.04% TOOS
Test solution: A 0.3 mM ZFL solution with FV added to a final concentration of 0, 0.1, 0.2, 0.3 mM.
After pre-warming 0.5 ml of Reaction Solution 1 at 37 ° C. for 5 minutes, 0.05 ml of the above test solution was added, and 37 ml
The reaction is carried out at 5 ° C. for 5 minutes, 0.5 ml of the reaction solution 2 is added, and after 5 minutes, the absorbance at 555 nm is measured. For the blank, use distilled water instead of the test solution. As a control, the same operation was performed using a reaction solution to which FOD-V of reaction solution 1 was not added.
The white circles in FIG. 8 indicate the measured values when FOD-V is not added, and the white squares indicate the values when FOD-V is added.

As is clear from FIG. 8 by using FOD-V and FOD, FZ-V was able to quantitatively measure FV after erasing ZFL in the test solution.
Sequences obtained by converting the 372nd lysine of the amino acid sequence 372 of the sequence listing into tryptophan, methionine and valine are shown in sequence listing sequence numbers 2, 3 and 4, respectively.

<Measurement of glycated albumin ratio>
R-1 Proteolytic reagent
50 mM POPSO acid buffer (Wako Pure Chemical Industries, Ltd.) pH 7.5
2500U / ml protease type XXIV (manufactured by Sigma)
1% Sulfuric acid-3-[(Coleamidopropyl) dimethylammonio] -2-hydroxy-
1-Propane (Sigma)
5U / ml ascorbate oxidase (Roche)
5% DMSO
5 mM 4-aminoantipyrine
R-2 Reagent for measuring glycated amino acid
150 mM HEPES acid buffer (Wako Pure Chemical Industries, Ltd.) pH 7.5
5mM TODB
10U / ml POD
20U / ml R-FOD-II
3% glutamic acid
R-3 Albumin pretreatment reagent
10 mM Tris-HCl buffer pH 8.0
0.3% sodium lauryl sulfate
R-4 Albumin coloring reagent
200mM succinate buffer (Wako Pure Chemical Industries, Ltd.) pH5.5
0.15mM Bromocresol Purple (Wako Pure Chemical Industries, Ltd.)
0.3% Tx-100 (Wako Pure Chemical Industries)
<Sample>
1: 35 samples of healthy and diabetic serum each
2: Control serum H (manufactured by BML) was used as a calibrator.
Glycated albumin concentration of the calibrator measurement HPLC methods measurements and enzymatic method of clinical specimens is preset to match. The albumin value was shifted from that of CRM470.
<Reaction procedure>
Incubated with R-1 to 37 ° C., was added to the sample 8μl to 240 .mu.l, to start the reaction at 37 ℃, R-2 after exactly 5 minutes; it was added 80 [mu] l. Changes in absorbance at 555 nm were measured before addition of R-2 and 5 minutes after the addition. Separately, control serum H and distilled water were measured to prepare a calibration curve, and the glycated albumin concentration in the sample was calculated.
On the other hand, R-3 incubated at 37 ° C .; albumin pretreatment reagent; 2 μl of the sample was added to 160 μl, and the reaction was started at 37 ° C. After exactly 5 minutes, R-4; albumin coloring reagent; 160 μl was added. Absorbance at 600 nm was measured before and 5 minutes after addition of the albumin coloring reagent. In addition, a calibration curve was prepared from a sample having a known concentration of distilled water and albumin instead of the sample, and an albumin measurement value was obtained.
GA% by the enzyme method was determined from GA% = GA concentration / albumin concentration × 100. Moreover, the HPLC method measured value was measured using Hi-Auto G.A.A 2000 (made by Arkray). The results are shown in FIG.

As can be seen from FIG. 9, the enzymatic method and the HPLC method showed a very good correlation with r = 0.998. Moreover, even when all of the reagents were stored in a liquid state at 37 ° C. for 2 weeks, the performance did not change. From this, 1) avoiding the effects of globulin components and ascorbic acid, 2) stabilizing proteases and enzymes that act at least on glycated amino acids, 3) accurately measuring albumin, 4) avoiding the effects of glycated hemoglobin It was clear that glycated albumin was measured and glycated albumin ratio was measured.

<Composition containing at least an enzyme that acts on glycated amino acids in the first reagent and a protease in the second reagent>
R-1
200 mM POPSO buffer pH 7.5
5 mM 4-aminoantipyrine
10 U / ml POD
20 U / ml R-FOD
5 U / ml ascorbate oxidase
3% glutamic acid
R-2
20 mM piverazine-1,4-bis (2-ethanesulfonic acid) buffer pH 6.5
20% DMSO
8000 U / ml protease type XXIV
4% Sulfuric acid-3-[(Coleamidopropyl) dimethylammonio] -2 hydroxy-
1-propane
5 mM TODB
R-3,4 Same as Example 22 <Sample> Same sample as Example 22 and 10-200 μM FZL
<Reaction Procedure> The same results as in Example 22 are shown in FIG.

As can be seen from FIG. 10, even when an enzyme that acts on at least a glycated amino acid in the first reagent and a protease is formulated in the second reagent, the glycated protein can be measured well in a short time of 10 minutes. Even when a glycated amino acid is present, it is clear that the glycated amino acid is erased by an enzyme acting on at least the glycated amino acid prescribed in R-1, and the glycated protein can be measured more accurately.
The correlation between this reagent and the HPLC method is
Enzymatic method GA% = 1.03 * HPLC method GA% -0.3 R = 0.99 showed good correlation, and it was also clear that glycated protein was accurately measured. Furthermore, the reagent was in a liquid state and its performance was not deteriorated even when stored at 37 ° C. for 3 weeks or refrigerated for 15 months.
[Reference to deposited biological materials]

(1) Name and address of the depository institution that deposited the biological material
National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center
1st, 1st East, Tsukuba City, Ibaraki, Japan 1st 6th (Zip 305-8566)
Date of deposit to Loi's institution (original deposit date)
January 16, 2001 Deposit number assigned to the institution of High
FERM BP-7847
(2) Name and address of the depository institution that deposited the biological material
National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center
1st, 1st East, Tsukuba City, Ibaraki, Japan 1st 6th (Zip 305-8566)
Date of deposit to Loi's institution (original deposit date)
January 16, 2001 Deposit number assigned to the institution of High
FERM BP-7848

According to the present invention, it has become possible to more accurately measure the ratio of glycated protein and glycated albumin in a subject. Therefore, it can be usefully used as a clinical test drug.

It is a graph which shows the measurement curve and reproducibility of the HSA substrate solution (4g / dl) based on Example 4 of this invention, a gamma globulin, and the globulin IV substrate solution. It is a graph which shows the measurement curve and reproducibility of the Hb substrate solution (4g / dl) based on Example 5 of this invention, a gamma globulin, and the globulin IV substrate solution. It is a graph which shows the measurement curve of glycated albumin based on Example 6 of this invention. It is a graph which shows the effect of the kind of buffer agent on stabilization of ascorbate oxidase in the composition for glycated protein measurement based on Example 9 of this invention. It is a graph which shows the effect of the kind of stabilizer which acts on stabilization of the protease based on Example 11 of this invention. It is a graph which shows the effect of the kind of stabilizer which acts on stabilization of the enzyme which acts on at least glycated amino acid based on Example 12 of this invention. The common structure from the plasmid pcmFOD1 of Example 20 of this invention to pcmFOD5 is shown. It is a graph which shows the measurement result of the light absorbency of 555 nm in the glycated valine density | concentration reaction liquid which erase | eliminated mixed glycated lysine by this mutant fructosyl amino acid oxidase of Example 21 of this invention, and a non-erased reaction liquid. It is a graph which shows the correlation of the enzyme method and HPLC method of the glycoalbumin ratio measurement result based on Example 22 of this invention. It is a reaction curve of the glycated protein measuring reagent based on Example 23 of this invention.

Claims (1)

In a composition for measuring a glycated protein using a protease and an enzyme that acts at least on a glycated amino acid, protease and 1) deoxycholic acid, deoxycholic acid amide, cholic acid amide, octyl glucoside, quaternary ammonium salt, quaternary One or more selected from ammonium salt type cationic surfactants, concanavalin A or betaine,
And / or 2) a buffer having no ascorbate oxidase and 4- (2-hydroxyethyl) -1-piperazinyl group,
A composition for measuring a glycated protein characterized by coexisting sucrose.